White blood cells called T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. In the unactivated state, these cells circulate in the blood and accumulate in lymphoid tissues such as lymph nodes and spleen. Upon encounter with foreign materials (antigens) on the membranes of specialized antigen presenting cells (dendritic cells), these resting T-cells become activated, undergo numerous cell divisions, and differentiate into effector cells. The effector cells leave the lymphoid tissues and blood, entering sites of infection to combat pathogens. They can also invade normal tissues where their activity can cause autoimmune pathology. After elimination of an infecting organism, most of the activated T-cells die, but some remain as memory cells, to provide a more rapid and vigorous response if the same pathogen is encountered in the future. Recent work has suggested that this general scheme applies to both CD4 and CD8 T-cells, but that there are also important differences in the signals that control the extent of proliferation and the survival of memory cells for these two T-cell lineages. Furthermore, there are also data suggesting that memory cells may be of more than one type, with some recirculating in lymphoid compartments and others patrolling peripheral tissues. The former may provide the major source of new cells upon re-infection, whereas the latter may mediate the earliest effector response to the infection. The proper balance of both types may be critical for effective T-cell mediated host defense. Other lymphocytes such as NK cells and regulatory T cells contribute to both the enhancement and suppression of these T cell responses through direct and indirect means. This project attempts to gain both a qualitative (especially tissue-specific 4 dimensional space and time) and a quantitative understanding of the activation, differentiation, migration, cell-cell interaction, memory status, and reactivation properties of both CD4 and CD8 T-cells. Issues such as the route, amount, and frequency of antigen exposure, as well as the presence or absence of adjuvants that stimulate the innate immune system, are being studied for their effects on the generation and tissue distribution of effector and memory CD4 and CD8 T-cells. The movement of activated T-cells into non-lymphoid tissues is being analyzed using both conventional cellular immunological methods and newer imaging techniques that allow high resolution dynamic observation of how cells migrate, interact, and carry out their effector functions. Through this research, a better understanding of lymphocyte dynamics during an immune response to infection or after vaccination or during an autoimmune response will be established. These new insights can contribute to the more effective design of vaccines and to strategies for the amelioration of autoimmune processes. We have established a robust system for vaccination using the non-replicating pox vector MVA, to permit in situ analysis of immune cell behavior in response to a clinically used vaccine vector. Variants of the virus have been developed that encode both fluorescent proteins and model antigens that are recognized by TCR transgenic T cells expressing distinct fluorescent proteins. This allows for tracking of the sites of viral infection and of the location and dynamic behavior of antigen-specific T cells during immune responses in situ, using our advanced 2-photon intravital imaging methods. Preliminary studies have suggested unexpected locations for some of the cells initially infected by MVA within draining lymph nodes, potentially distinct cells involved in initial antigen presentation to CD4 T cells vs. CD8 T cells, the rapid loss of directly infected cells, a role for cross-presentation as well as direct presentation in the activation of CD8 T cells, and different locations of naive vs. memory CD8 T cells in the lymph node. In the case of the distinct sites of CD4 and CD8 T cell antigen engagement, we are now trying to understand how what appears to be dispersed initial antigen activation of these two cell types on different antigen-presenting cells can be reconciled with data from other laboratories and from the LBS that indicate a requirement for antigen co-presentation to the CD8 and CD4 T cells on the same dendritic cells for induction of robust immune memory. We have also utilized this model for examine the effect of regulatory T cells (Tregs) on the MVA-induced response and have obtained data showing a striking differential effect of these cells on CD8 T cells, especially with respect the balance of highly polarized effectors versus multifunctional/ memory T cells emerging after priming. We have dissected the mechanism of the suppression of effector generation by Tregs, revealing a key role for limitation in the late (post-activation) availability of the cytokine IL-2. In other experiments building on a combination of our highly multiplexed immunofluorescence imaging and our 2-photon intravital methods, we have accumulated data on the delivery of antigen from subcutaneous sites to the draining lymph node and the role of distinct subpopulations of macrophages and dendritic cells in acquiring the antigen in different regions of the lymph node. With respect to effector function, a combination of intravital imaging and flow cytometry studies in a model of delayed-type hypersensitivity have revealed that effector cells in an inflamed, not tolerogenic, site undergo a single round of activation to cytokine secretion status in concert with an arrest of migration, followed by a loss of cytokine production as the cells mobilize from the antigen presenting cell and resume rapid movement in the tissue that lasts as long as we can image (>10 hours). These data imply a mechanism of tuning of effector cells upon initial activation in the tissue that limits damaging cytokine production if the antigen load is not increasing, and also suggests that eventual elimination of a pathogen will depend on a constant influx of new effectors from secondary lymphoid tissue. Preliminary data suggest that a negative feedback loop involving regulatory surface proteins whose expression increases shortly after antigen stimulation of effector T cells in the tissue depresses signaling through the TCR and hence, desensitizes the T cells with respect to the available antigen level. We are continuing our studies of activated T cell help for humoral antibody responses from B cells, with a focus on the generation and behavior of follicular helper CD4 T cells (Tfh). New reporter systems are being developed to track these cells and to characterize their dynamic and functional interactions with B cells in the interfollicular region and especially within germinal centers. Progress has been made in the initial steps of constructing and validating a novel reporter of Bcl6 protein expression (rather than gene transcription). Finally, we have developed new data suggesting that the in vitro paradigm of autocrine cytokine-induced polarization of CD4+ T cells is an oversimplification of the in vivo reality. In particular, both in vitro and in vivo, under suitable conditions, Th1 and Th2 effector T cells can be generated in the absence of the canonical cytokine (IFNgamma and IL-4, respectively), or the relevant STAT protein. A key determinant of the fate of the T cells is the strength of signaling through the T cell antigen receptor, and in vivo, preliminary data suggest that changes to dendritic cell morphology and capacity for cell-cell interactions plays an important role in determining the strength of signaling to T cells and their ultimate polarized fate.